Role of Estrogen Receptor in Endometrial Cancer Initiation and Progression Epidemiologic studies dating back thirty years demonstrated that activation of the estrogen receptor (ER) signaling pathway by unopposed estrogen is a risk factor for the formation of endometrial carcinoma. Despite this observation, critical questions remain unanswered. (1) Why do only subsets, and not all women, exposed to unopposed estrogen develop endometrial cancer? (2) What are the ER mediated mechanisms that can lead to the formation of endometrial cancer? (3) Where in the uterus are estrogenic effects mediated, endometrial epithelium and/or stroma? A major limitation in studying endometrial cancer has been a paucity of mouse models, predominantly due to a lack of endometrial epithelial cell-specific promoters. Recently we have had a breakthrough in my laboratory with the establishment of an in vivo endometrial regeneration model from dissociated populations of endometrial epithelium and stroma. This system can be used to assay two important biological parameters in isolated subpopulations of endometrial cells: response to cell autonomous and paracrine oncogenic stimuli. Cell autonomous activation of the PI3Kinase axis via deletion of PTEN or paracrine signaling by growth factors are two pathways implicated in the initiation and progression of endometrial cancer, but mechanisms through which each of these pathways can interact with estrogen receptor signaling has not been determined. While ER 1 and ER 2 are both expressed in the endometrium, based on functional studies ER 1 - the main focus of the outlined studies in this grant - is thought to be the predominant form of ER in this tissue. In this proposal, we plan to delineate the epithelial specific role of ER 1 in cooperation with cell autonomous and paracrine oncogenic signals in endometrial cancer. We hypothesize that activation of epithelial ER 1 can cooperate with cell autonomous signals such as PI3Kinase pathway activation. Signaling through ER 1 may be essential for the initiation of endometrial hyperplasia and cancer mediated by cell autonomous or paracrine oncogenic signals. Oncogenic paracrine growth factor signals may lead to ligand independent activation of ER 1, which may be a critical pathway for the progression of endometrial cancer to a hormone refractory state. Our specific aims are (1) Examine potential synergy between the activation of the PI3Kinase and the ER 1 signaling axes in the endometrium. Endometrial epithelial cells harvested from PTENloxP/loxP mice will be lentivirally infected with ER 1 and Cre- recombinase resulting in deletion of PTEN and over expression of ER 1. These cells will be placed in the in vivo endometrial regeneration model and their tumor forming capacity will be compared to controls. (2) Assess whether epithelial ER 1 is essential for the formation of endometrial hyperplasia or cancer. Using a Cre-loxP recombination system, ER 1 will be deleted in endometrial epithelia harvested from floxed ER 1 mice and their response to paracrine or cell autonomous oncogenic signals will be assessed. (3) Determine the role of paracrine growth factor signaling in the ligand dependent and independent activation of ER 1. Retroviral delivery vectors will be used for the expression of candidate growth factors in endometrial stromal cells. Growth factor expressing stromal cells will be recombined with adult dissociated endometrial epithelia and placed in the in vivo endometrial regeneration model. Western blot and immunohistochemistry will be used to assess activation of ER 1 signaling in control and growth factor over expressing regenerated grafts. Findings in this proposal can define the epithelial specific role of ER 1 signaling in the initiation and progression of endometrial cancer in conjunction with cell autonomous and paracrine oncogenic signals. Our findings could have critical implications for the subset of patients with endometrial tumors driven by the ER 1 signaling, in whom ER 1 could be a valid therapeutic target. Results of this proposal could be a step toward individualizing therapy for this common gynecologic malignancy in the growing population of female veterans.